A dose-response characterization of transcranial magnetic stimulation intensity and evoked potential amplitude in the dorsolateral prefrontal cortex

By combining transcranial magnetic stimulation (TMS) with electroencephalography, human cortical circuits can be directly interrogated. The resulting electrical trace contains TMS-evoked potential (TEP) components, and it is not known whether the amplitudes of these components are stimulus intensity dependent. We examined this in the left dorsolateral prefrontal cortex in nineteen healthy adult participants and extracted TEP amplitudes for the N40, P60, N120, and P200 components at 110%, 120%, and 130% of resting motor threshold (RMT). To probe plasticity of putative stimulus intensity dose-response relationships, this was repeated after participants received intermittent theta burst stimulation (iTBS; 600 pulses, 80% RMT). The amplitude of the N120 and P200 components exhibited a stimulus intensity dose-response relationship, however the N40 and P60 components did not. After iTBS, the N40 and P60 components continued to exhibit a lack of stimulus intensity dose-dependency, and the P200 dose-response was unchanged. In the N120 component, however, we saw evidence of change within the stimulus intensity dose-dependent relationship characterized by a decrease in absolute peak amplitudes at lower stimulus intensities. These data suggest that TEP components have heterogeneous dose-response relationships, with implications for standardizing and harmonizing methods across experiments. Moreover, the selective modification of the N120 dose-response relationship may provide a novel marker for iTBS plasticity in health and disease.


Procedure
EEG data were collected concurrently with TMS delivery at three different stimulus intensities, 110%, 120%, and 130% RMT, before and after a course of iTBS (described below in Section "TMS").These stimulus intensities reflect suprathreshold increments (relative to motor threshold) abutting the upper bound of stimulator output for most participants with our TMS-EEG configuration.This is consistent with TMS-electromyography (TMS-EMG) techniques where dose-response characterizations are more common.During TMS-EEG recordings, participants kept their eyes focused on a fixation point.To reduce auditory neural responses to the TMS coil discharge, participants wore ear plugs and noise-cancelling headphones playing a white noise recording.A memory foam pad compressible to 2 mm was placed on the coil to maximize comfort and minimize bone conduction.Each testing session lasted approximately 2 h.See Fig. 1a for a schematic diagram of the study procedure.TMS TMS was delivered using biphasic pulses with a MagPro X100 stimulator (MagVenture) and a Cool-B70 figureeight coil held tangentially to the head and positioned 45 degrees laterally from the midline.RMT was determined with motor evoked potentials (MEPs) using an electromyographic electrode placed over the right first dorsal interosseous (FDI) muscle and a reference electrode placed over the first metacarpophalangeal joint.After locating the left motor cortex first dorsal interosseous muscle hotspot, RMT was defined as the minimum stimulus intensity required to generate MEPs at ≥ 0.05 mV on at least five of ten stimulations.
TMS was delivered to the left DLPFC defined using the Beam F3 method 31 and the same location was used for both single-pulse TMS and iTBS.To ensure fidelity and consistency of coil placement across the experiment, Neuronavigation was utilized with a template brain registered to the participant's anatomy (Visor2, ANT Neuro).

EEG
Continuous EEG data were collected using a BrainVision actiCHamp Plus system (Brain Products GmbH; Gilching, Germany) from an array of 32 TMS-compatible active slim electrodes (10-20 positioning; Cz as reference) at a rate of 10 kHz.DC-coupling of the amplifier was used to avoid amplifier saturation form the TMS artifact.Impedances of ≤ 10 kΩ were established prior to testing.data from the full epochs were re-referenced to the common average, baseline corrected (− 100 to 0 ms), and deconcatenated into separate conditions.See Supplementary Table S1 for information on trials, channels, and components removed for each condition.

TMS-evoked potential amplitudes
To assess the local response to TMS over the left DLPFC, our region of interest (ROI) was the average of four electrodes around the stimulation site (F3, FC5, FC1, C3).We focused on four components within the following time windows: the N40 (20-55 ms), P60 (40-90 ms), N120 (80-150 ms), and P200 (150-250 ms).Trials were averaged within each condition and TMS-evoked potential (TEP) amplitudes were calculated by averaging data ± 5 ms around the identified peak.As broad windows were used for finding peaks, each individual trace was manually checked before acceptance of peak latency and amplitude values.See Fig. 1b & c for a representative TEP trace and the topographic location of the electrodes included in the ROI, respectively.

Statistics
Statistical analyses were carried out in IBM SPSS Statistics Version 25.To evaluate the dose-response function at the three stimulus intensities, separate general linear models (GLM) were conducted for each component using TEP amplitudes before iTBS with stimulus intensity (110%, 120%, 130%) as the within-subjects factor.General linear mixed effects models (GLMEM) with within-subjects intercepts were conducted for each component to evaluate changes in the dose-response function following iTBS, with intensity (110%, 120%, 130%) and timepoint (pre-iTBS, post-iTBS) as within-subjects factors.Post-hoc tests included pairwise parameter estimation for significant main effects and paired t-tests for simple main effects in significant interactions.Bonferroni corrections were applied to correct for multiple comparisons.

Results
Demographic data are presented in Table 1.All four components of interest (N40, P60, N120, P200) were reliability elicited in all conditions, with latency values presented in Table 2, individual datapoints are presented in Supplementary Figs.S1 & S2, and topographic maps for grand average components are presented in Supplementary Fig. S3.TEP component peaks were not windowed based on the grand average because the latencies for these peaks were variable across individuals but highly consistent within individuals (Intra-class correlations: N40 = 0.84, P60 = 0.91, N120 = 0.92, and P200 = 0.84), consistent with considerable variability in the structure and function of the prefrontal cortex.Although the P30 component is commonly observed in TEP investigations of cortical excitability 34 , the magnitude of the TMS artifact at higher intensities impacted the reliability of this component and we excluded the P30 from analyses as it was not present in all conditions.As peak identification parameters (described in Section "TMS-evoked potential amplitudes") did not always identify local peaks at every tested electrode in every participant, missing amplitudes were replaced using values from 15 unique datasets generated via multiple imputation (maximum 200 iterations, fully conditional specification, linear regression).In total, 3.95% of component amplitudes were not identified within the pre-defined windows, and Little's MCAR test suggested that these values were missing at random (p > 0.999).For datasets with missing values, the median

Discussion
In this study, we evaluated TEP component stimulus intensity dose-response relationships in the left DLPFC using three stimulus intensities and probed plasticity within these putative dose-response relationships.Contrary to our hypotheses, our analyses suggest that stimulus intensity dose-response relationships are not generalized in the DLPFC, and that increasing stimulus intensity selectively impacts later TEP components.Importantly, it is only a subset of TEP components, those with a pre-existing stimulus intensity dose-response relationship, that appear to be subsequently modifiable by iTBS.We observed significant stimulus intensity dose-response relationships in the N120 and P200 components and, surprisingly, only the N120 dose-response relationship was modified by iTBS, suggesting it is sensitive to both the effects of varying intensity in single-pulse protocols and plasticity-inducing protocols.However, when the interest is on earlier TEP components, our data suggest that component amplitudes are consistent and reliable at lower stimulus intensities such that higher intensity TMS pulses, and their associated TMS-induced artifact, may not be necessary.Moreover, our data have important implications for the standardization of TMS-EEG stimulation parameters, particularly for the valid interpretation of the N120 and P200 peaks, and for the interpretation and synthesis of data with different methodologies.
TMS-evoked potentials in the DLPFC are canonically the N40, P60, N120 and the P200.Pharmacological studies have revealed different contributions to each of these, with the N40 representing an integration of GABA-A and NMDA receptor contributions 35,36 , the P60 reflecting glutamatergic AMPA receptor signalling 35 , the N120 reflecting GABA-A and GABA-B receptor signalling [35][36][37][38][39] and the P200 reflecting the integration of multiple inputs as a global marker of excitability 40,41 .Accordingly, our data suggests that input-output functions in the DLPFC may be particularly relevant for the study of GABAergic signalling and plasticity.
The N120 component is also of clinical interest and has been implicated in neuropsychiatric conditions.For example, Noda and colleagues demonstrated that N120 amplitudes were significantly smaller in participants with schizophrenia compared with healthy controls 22 .Tallus and colleagues found that N120 amplitudes were significantly higher in symptomatic compared to asymptomatic mild traumatic brain injury patients and controls 42 .In a machine learning paradigm, Zhang and colleagues found that the N120 component was able to differentiate cognitively impaired participants from healthy controls with relatively high accuracy 17 .The N120 also plays a notable role in major depressive disorder, with Voineskos and colleagues demonstrating that amplitudes are significantly larger in adults with depression compared with healthy controls 21 , and Dhami and colleagues reproducing this effect in depressed youth 20 .
The N120 is also amenable to change with both single-session and long-term repetitive TMS protocols.For example, a 6-week randomized controlled trial of repetitive TMS over the left DLPFC in treatment-resistant depression resulted in decreased N120 amplitudes following active stimulation 28 .Chung and colleagues have demonstrated significant modulation of N120 amplitudes following iTBS over the left DLPFC in three different studies [24][25][26] .This effect of iTBS on N120 amplitudes has also been observed in primary motor cortex N120 43 .The implication of a stimulus intensity dose-response relationship for this GABAergic marker is very relevant, as GABAergic plasticity is hypothesized to underlie large scale network changes with rTMS 44 .Further, the iTBS protocol is thought to induce NMDA receptor dependent plasticity in part through GABA autoreceptor activation at the theta frequency 45,46 .Our data suggest that future studies examining the N120 component may benefit from incorporating stimulus intensity dose-response characterizations to maximize the interpretability of findings and the prognostic utility in therapeutic contexts.Further, we believe it is important to highlight the strength of the dose-response approach when characterizing cortical function, as it can reveal a shift in dose-response relationships whereas single intensities may not show the neuroplastic effects of plasticity protocols such as iTBS.We caution against interpreting that any one stimulus intensity is optimally suited to uncovering these effects based on statistical separation in post-hoc testing.
An important caveat to our work is the narrow range of stimulus intensities used, all of which were suprathreshold to an FDI motor threshold.It is possible that stimulus response relationships in the prefrontal cortex are observed at intensities that are inferior to motor threshold in earlier TEP components, in which case our data would instead reflect a ceiling effect once the neural activation threshold of 100 V/m is met 47 .This may be consistent with the sigmoidal function that emerges from TMS-EMG, where motor cortex neurons are recruited by an increasing stimulus intensity until a limit wherein expanding the volume of stimulated cortex no longer recruits additional neurons involved in the motor representation and an MEP plateau is reached 48 .While it is possible that DLPFC early components exhibit a dose-response that is saturated at suprathreshold intensities, in motor cortex subthreshold TMS pulses only elicit weak TEP waveforms 49 .We cannot exclude the possibility, however, that subthreshold pulses would reveal a stimulus-intensity dose-response in the DLPFC.There is considerable evidence that properties of sigmoidal TMS-EMG input-output curves are sensitive to iTBS effects [50][51][52][53] .Therefore, future research should evaluate the full range of TEP properties in DLPFC to determine whether a similar sigmoidal function is present and its practical applicability to plasticity-inducing protocols.

Limitations
The auditory evoked potential typically manifests 100-200 ms post-stimulation, and despite the steps taken to mitigate this confound (i.e., ear plugs, noise-cancelling headphones playing white noise, and a foam pad placed between the coil and head), we cannot exclude the possibility that auditory effects contributed to our stimulus intensity dose-response findings [54][55][56][57] .It is also worth noting that we did not confirm effectiveness by asking participants if they could hear the coil discharge.
We were unable to reliably examine the P30 component, and the N40 and P60 peaks were small in amplitude and may have benefited from more trials to improve signal-to-noise ratio and peak estimation.However, the validity of a within-individual design including a pre-post iTBS comparison required balancing experimental We determined stimulation intensity relative to first dorsal osseous RMT.However, we acknowledge that anatomical differences between prefrontal cortex and motor cortex (including but not limited to scalp-to-cortex difference and gyral configuration) introduce important structural and functional variability across participants.However, this is mitigated in part by the use of a within-subject design.
Lastly, informed by TMS-EEG dose-response relationship in motor cortex 49 , we did not include stimulus intensities subthreshold to FDI motor threshold, but it is possible that this precluded detecting stimulus intensity dose-response relationships in the N40 and P60 components.

Conclusion
Using TMS-EEG to probe stimulus response relationships in the DLPFC, we show that TEP components do not all exhibit dose-response relationships to suprathreshold stimulation.Furthermore, our data suggest that neurostimulation, in this case iTBS, produces a selective shift in the stimulus intensity dose-response function only for the N120 component.Component amplitudes elicited at a single intensity should be interpreted with caution, and future studies of GABAergic signalling with TMS-EEG would benefit from incorporating stimulus intensity dose-response characterizations.

Figure 1 .
Figure 1.Diagram of the study procedure, region of interest electrodes, and illustrative TMS-evoked potential traces.(a) Schematic diagram of the study procedure.After determination of resting motor threshold (RMT), concurrent single-pulse transcranial magnetic stimulation and electroencephalography (TMS-EEG) was conducted at three different stimulus intensities (110%, 120%, and 130% RMT) before and after intermittent theta burst stimulation (iTBS) over the left dorsolateral prefrontal cortex.(b) TMS-evoked potentials from the average of the 120% RMT condition at baseline with components labelled.The vertical line at time = 0 marks the onset of the TMS pulse.The shaded grey bar represents the interpolated region (− 2 to 15 ms).The traces of each individual electrode are shown in light grey.The region of interest trace is superimposed in blue.c) Scalp topographic map with electrodes labelled.The average signal from the four circled electrodes (F3, FC5, FC1, C3) constitute the region of interest.Image generated using EEGLAB v14.1.1 (https:// sccn.ucsd.edu/ eeglab/ downl oad.php).

Figure 2 .
Figure 2.Grand average TMS-evoked potential traces and dose-response functions before intermittent theta burst stimulation.Grand average (N = 19 participants) TMS-evoked potential traces at the region of interest (F3, FC5, FC1, C3) and associated dose-response functions for the (a) N40, (b) P60, (c) N120 † , and (d) P200 TMSevoked potential components before intermittent theta burst stimulation over the left dorsolateral prefrontal cortex.It is important to note that grand average traces do not directly inform mean amplitudes as these are extracted on a per participant basis with variability in peak latency.Error bars represent ± 1 standard error of the mean.P-values correspond to general linear models.*p < .05. **p < .01. † One outlier was detected in the 130% condition but data quality did not justify exclusion from analyses.

Figure 3 .
Figure 3.Grand average TMS-evoked potential traces and dose-response functions before and after intermittent theta burst stimulation.(a) Grand average (N = 19 participants) TMS-evoked potential traces at the region of interest (F3, FC5, FC1, C3) for each stimulus intensity (110%, 120%, 130% RMT) before and after intermittent theta burst stimulation over the left dorsolateral prefrontal cortex.(b-e) dose-response functions for the N40, P60, N120, and P200 TMS-evoked potential components, respectively, before and after intermittent theta burst stimulation over the left dorsolateral prefrontal cortex.It is important to note that grand average traces do not directly inform mean amplitudes as these are extracted on a per participant basis with variability in peak latency.Error bars represent ± 1 standard error of the mean.P-values correspond to general linear mixed effects models.*p < .05. **p < .01.
https://doi.org/10.1038/s41598-023-45730-ywww.nature.com/scientificreports/length and the possibility of participant movement.Despite the use of neuronavigation to ensure accuracy and consistency of placement, subtle shifts would have compromised the interpretation of stimulation effects.

Table 2 .
Latency -value generated by the imputation procedure is reported.See Supplementary TableS2for outcomes of analyses with original and imputed datasets.Statistical significance of the effects described below were equivalent with the original and imputed datasets.